Antigen presentation by MHC class I molecules to CD8 T cells is a major pathway by which the acquired immune system detects and eliminates virus infected cells. All nucleated cells express MHC class I molecules and are thus potentially capable of direct antigen presentation to CD8 T cells upon infection. However, several, but not all, recent studies suggest that predominantly DCs (or a specific subset of DCs) are uniquely required for in vivo priming of CD8 T cell to virus. Because pathogens may not directly infect these requisite DCs, cross presentation pathways have been proposed;in essence, the infected cell may not be the primary antigen presenting cells. Additionally, for many arthropod-transmitted viruses, virus-specific antigens may require transfer from migratory DCs in the skin to lymph node resident DCs to efficiently prime CD8 T cells. However, the mechanism by which pathogen-specific antigens are shuttled from the infected cells to DCs or between DC subsets is unknown. Not surprisingly, these same issues of direct presentation vs cross-presentation also apply to CD8 T cell responses to tumors or following DNA vaccination. The experiments proposed in this application will address outstanding questions of the molecular and cellular basis of in vivo priming of CD8 T cells against viruses and following DNA vaccination. As unique probes of antigen presentation pathways in virus immunity, we have engineered MHC class I molecules as peptide-preloaded single chains (or SCTs), defined immunodominant epitopes detected by CD8 T cells after West Nile virus (WNV) infection, and characterized novel T cell receptor mimic monoclonal antibodies that recognize MHC class I loaded with WNV peptides. These reagents will be used to dissect the cellular and immunologic mechanisms by which protective CD8 T cell responses against WNV infection are generated.